Peptides used as agonists and/or inhibitors of amyloid formation and cytotoxicity and also for use in alzheimer&#39;s disease, in type ii diabetes mellitus and in spongiform encephalophathies

ABSTRACT

Certain peptide molecules can be used as the basic structures (template molecules) for inhibiting and analysing amyloid formation and cytotoxicity in amyloid illnesses. These peptides have an effect on the molecules which are responsible for the amyloid illnesses (for their part amyloid-forming peptides and proteins). The peptides are thus either inhibitors themselves or agonists of amyloid formation and cytotoxicity or can serve as a template for identifying and producing further inhibitors and agonists and can be used as molecular tools during analysis.  
     The peptide molecules have generally 3-15 amino acids, and preferably a maximum of 10 amino acids, and at least an active peptide sequence GA, preferably GAI, and even more preferably one selected from the group consisting of GAIL, FGAIL, NFGAIL, NNFGAIL, SNNFGAIL, NFGAILSS and SNNFGAILSS, or the group consisting of GAII, KGAII, NKGAII, SNKGAII, GSNKGAII, NKGAIIGL and GSNKGAIIGL, or the group consisting of AGAVV, AAGAVV, VYYGAVV, HVAAGAVV, AAGAWGG and HVAAGAVVGG. The peptide sequence generally has at least one hydrogen molecule, and preferably every second hydrogen molecule, of an amide bond replaced by a methyl group.

[0001] The invention relates to peptides with 3-15 amino acids whichfunction as agonists and/or inhibitors in amyloid formation and/ortoxicity and which can be therefore used in various related clinicalpictures.

[0002] New strategies and active agents for the therapy and diagnosis ofthe above-mentioned amyloid illnesses are being researched world-wide.However, there is still no means of treating these illnesses withmedicines/pharmaceuticals. According to predominant expert opinion,certain amyloid proteins, which are specific to each illness, arecausally responsible for the occurrence of amyloid illnesses because oftheir amyloid genesis or aggregation. The mechanism for amyloid genesisand the associated cell death (cytotoxicity) in these illnesses iswidely unknown and correspondingly, highly specific inhibitors havehence not been identified. Pharmaceuticals for treating amyloidillnesses on the basis of such inhibitors have therefore also not beendeveloped.

[0003] In exactly the same way, the protein-chemical,technical-analytical problems, which are caused by amyloid formation,(formation of insoluble protein aggregates, so-called amyloidstructures) have up to now not permitted the analysis of amyloidformation. This has contributed to the fact that the mechanism foramyloid formation is still widely unexplained. For analysing theformation of amyloids, constructive diagnostic methods (fast in vitrotests for evaluating the amount, duration and quality of the amyloidstructures) still therefore do not exist either. For example, adiagnosis for Alzheimer's can only be performed symptomatically(increasing forgetfulness or similar) or post mortem. Reliable bloodtests, for example, cannot be performed during the lifetime of thepatient.

[0004] On this basis it is therefore the object of the invention topropose appropriate peptides, which can function as agonists and/orinhibitors of amyloid formation and/or cytotoxicity.

[0005] The object is resolved by the characterising features of claim 1.The sub-claims indicate advantageous further developments. Theapplication of the peptides is given in claims 7 to 9.

[0006] According to the invention, peptides with 3-15 amino acids areproposed, which contain at least the active sequence GA. It has beenshown, that these peptide molecules function as inhibitors and/oragonists of those amyloid peptides/proteins, which cause the amyloidillnesses Alzheimer's disease, Type II diabetes mellitus and spongiformencephalopathies (Creutzfeld Jacob disease, scrapie, BSE). The peptidemolecules according to the invention are in a position to inhibit theamyloid genesis or aggregation of the amyloid peptides/proteins ofamyloid peptide or _-AP (in Alzheimer's disease), amyline/IAPP (in TypeII diabetes mellitus) and prion protein (in spongiformencephalopathies). When inhibition of the amyloid genesis ofillness-inducing peptides/proteins is achieved, the cyto-toxic effect,which is caused by aggregation of _-AP, amyline/IAPP or prion protein,on tissue cells is inhibited.

[0007] The invention offers the following advantages relative to thestate of the art:

[0008] Simple chemosynthesis to a high degree of purity, and use,according to current methods, of fixed phase peptide synthesis (thepeptides described here are shorter, as a rule, than the potentialinhibitors described up till now and consist of only one type ofchemical component [=amino acids]).

[0009] high biological stability, which can be increased even more bythe simple chemical inclusion of unnatural amino acids.

[0010] broad biological and therefore potentially therapeuticapplicability (the high homology level between the correspondingsequences of amyloid-forming peptides/proteins makes possible theoverlapping application of inhibitors in all three illnesses).

[0011] few side-effects and little antigenicity when used as atherapeutic (the peptides, on the basis of their small size, have asmall tendency to induce immune reactions in the patient); otherinhibitor candidates which are described in the literature (see aboveantibodies or higher molecular serum components) are approx. 200-300times larger than the peptides which are described here.

[0012] high biological activity in vitro and thus high predictablebiological activity in vivo.

[0013] thus high predictability of therapeutic application.

[0014] It has been shown that, for the three areas of applicationdescribed at the beginning, various peptides respectively areparticularly appropriate, said peptides all having homology amongst oneanother (FIG. 1). The individual peptides for the three groups aredescribed subsequently in greater detail.

[0015] The appropriate peptide sequence, which is adequate for amyloidformation and cytotoxicity of IAPP, is the sequence FGAIL (one-lettercode) which comprises the amino acid residues 23-27 of IAPP. Lengtheningthis sequence in the direction of the N terminus of IAPP results equallyin small (<10 amino acid residues) peptide fragments which can formfibrils (an arranged aggregate structure, which is typical of amyloidillnesses) and has cytotoxicity. For the spontaneous aggregation, i.e.production of an oversaturated peptide solution of these peptides, aconcentration is required which is 100-100 times greater compared withIAPP. These sequences were successfully used as small molecularinhibitors of amyloid formation of IAPP, since they contain the shortestpeptide sequence and that which is necessary for the aggregation ofIAPP. Underlying this effect is the aggregation mechanism of IAPP and,in equal measure, other amyloid forming peptides (such as _-AP and theprion protein). The aggregation and amyloid formation results thus froman intermolecular _- folding leaf formation, for which intermolecular(between the molecular chains) hydrogen bridges and hydrophobicreciprocal effects are necessary between the side chains of certainamino acid residues. This _-folding leaf structure leads to anon-covalent bond firstly between two and subsequently between severalIAPP molecules which consequently form insoluble aggregates/amyloidstructures. The effect mechanism (inhibition) of the peptides accordingto the invention lies in bonding the aggregation-promoting,intermolecular reciprocal effects between two IAPP molecules. By meansof this, it has been achieved recently that the peptides themselves areinvolved in these reciprocal effects with IAPP. Thus competition(agonism) occurs between IAPP and the peptides for the free “bondingpoints” of IAPP. Their potential use as illness-diagnostics is alsobased on the agonistic effect of these peptides. Because of the factthat the soluble form of the peptides or lower concentrations(micromolar) neither aggregate nor have cytotoxicity, their applicationas inhibitors and Type II diabetes diagnostics is made possible.

[0016] The following peptide sequences are synthesised and used asinhibitors of amyloid formation: GAIL, FGAIL, NFGAIL, NNFGAIL, SNNFGAIL,NGGAILSS, SNNFGAILSS, the individual letters for the amino acids existaccording to the one letter code. Furthermore, H-molecules of the amidebond of the above peptide sequences are replaced by a methyl group, tobond the aggregation of peptides which is induced by_folding leafformation. The methyl groups were introduced at every second amide bond,however varying the number of methyl groupings. The peptide analoguesproduced contain a number of N-methyl substituted amide bonds, whichextend between one and half of the present amide bonds per molecule.Underlying the design of this class of aggregation inhibitors lies theidea that, by introducing the methyl group at every second amide bond,the dimerisation or the non-covalent bond of the small molecular peptidesequences of IAPP are not impaired; the otherwise consequentnon-covalent expansion of the_folding leaf structures, which leads toaggregation, is however comprehensively switched off. In the following,representative examples of this substance class which were usedsuccessfully as aggregation inhibitors of IAPP, are named:

[0017] (N-Me)-GA-(N-Me) IL, F(N-Me)-GA-(NME) IL, NF(N-Me)-GA-(N-Me)IL,

[0018] NNF(N-Me)-GA-(N-Me)IL, SNNF (N-Me)-GA-(N-Me)IL,

[0019] NF(N-Me)-GA-(N-Me)ILSS, SNNF (N-Me)-GA-(N-Me)-GA-(N-Me)IL,

[0020] G(N-Me)-AI(N-Me)-L, FG(N-Me)-AL(N-Me)-L, NFG(N-Me)-AI(N-Me)-L,

[0021] NNFG (N-Me) -AI(N-Me) -L, SNNFG(N-Me) -AI(N-Me)-L,

[0022] NFG(N-Me)AI(N-Me)-LSS, SNNFG(N-Me)-AI(N-Me)-LSS, FGA(N-Me)-IL,

[0023] NFGA(N-Me)-IL, NNFGA(N-Me) -IL, SNNFGA(N-Me) -IL,NFGA(N-Me)-ILSS,

[0024] SN(N-Me)-NFGAILSS, N-Me)-SN(N-Me)-NFGAILSS, (

[0025] N-Me)-SN(N-Me)-NF(N-Me)-GAILSS etc.

[0026] In the appropriate peptide, which is used for amyloid formationand cytotoxicity of _-AP, sequence regions of _-AP, which are foundbetween the amino acids 25 and 34, are of concern. In analogy to thesequence 23-27 of IAPP, the sequence of 28-32 (KGAII) in _-AP was foundto be the appropriate substance for forming amyloids and forneurotoxicity of the entire molecule _-AP. On the basis of the sameconsiderations as in the case of the IAPP amyloid forming inhibitors,the following were synthesised to IAPP homologue _-AP sequences and usedas inhibitors of _-AP amyloid formation and _-AP neuro-toxicity: GAII,KGAII, NKGAII, SNKGAII, GSNKGAII, NKGIIGL, GSNKGAIIGL. Furthermore, asis described above, analogues were also produced with substituted amidebonds. The structures of these analogues were designed according to thesame principle as the N-methylated inhibitors of IAPP amyloid formation(see above). Representative examples of this substance class are listedin the following:

[0027] (N-Me)GA-(N-Me)II, K(N-Me)-GA-(N-Me)II, NK(N-Me)-GA-(N-Me)II,

[0028] SNK(N-Me)-GA- (N-Me) II, GSNK(N-Me) -GA- (N-Me)II, NK(N-Me) -GA-(N-Me)IIGL,

[0029] GSNK(N-Me)-GA-(N-Me)II, G(NMe)AI(N-Me)-I, KG(N-Me)-AI(N-Me)-I,

[0030] NKG(N-Me)-AI(NMe)-I, SNKG(N-Me)-AI(N-Me)-I,GSNKG(N-Me)-AI(N-Me)-I,

[0031] NKG(N-Me)-AI(N-Me)-IGL, GSNKG(N-Me) -AI (N-Me)-IGL, KGA(N-Me)-II,

[0032] NKGA(N-Me)-II, SNKGA(N-Me)-II, GSNKG- A(N-Me)-IIGL,

[0033] GS(N-Me)-NKGAIIGL, (N-Me)-GS(N-Me) - NKGAIIGL,

[0034] (N-ME)-GS(N-Me)-GAIIGL etc.

[0035] The most suitable sequence, which is adequate for amyloidformation and cytotoxicity of PrP is AGAVV. This is to do with a partialsequence from the sequence 110-119 PrP. Further sequences for use asaggregation and toxicity inhibitors of PrP are GAIL, AAGAVV, VYYGAVV,HVAAGAVV, AAGAVVGG, HVAAGAVVGG. The corresponding N-methylated peptidesequences—analagous to the IAPP derivatives (see above)—are alsosuitable as aggregation and toxicity inhibitors. Some representativeexamples of N-methylated analogues are presented in the following:

[0036] (N-Me)-GA-(N-Me)VV, A(N-Me)-GA-(N-Me)VV, AA(N-Me)-GA-(N-Me)VV,

[0037] AAGA(N-Me)-VVGG, HV(N-Me)-AAGAVVGG, (N-Me)-HV(N-Me)-AAGAVVGG,

[0038] (N-Me)HV(N-Me)-AA(N-Me)-GAVVGG etc.

[0039] This invention is therefore concerned with peptide moleculeswhich can be used as the basic structures (template molecules) forinhibiting and analysing amyloid formation and cytotoxicity in amyloidillnesses. In this respect, these peptides have an effect on themolecules which are responsible for the amyloid illnesses (for theirpart amyloid-forming peptides and proteins). The peptides are thuseither inhibitors themselves or agonists of amyloid formation andcytotoxicity or can serve as a template for identifying and producingfurther inhibitors and agonists and can be used as molecular toolsduring analysis.

[0040] These peptides can be used as pharmaceutical inhibitors ofamyloid formation and cytotoxicity or as molecular tools for analysingamyloid formation and cytotoxicity in amyloid illnesses. Thus, this isto do with potential pharmaceuticals and analyses for the treatment anddiagnosis of the following illnesses which also occur in humans:

[0041] Alzheimer's disease

[0042] Type II diabetes mellitus

[0043] Spongiform encephalopathies

[0044] (e.g. Creutzfeld-Jacob disease, scrapie, BSE)

[0045] The diagnostic use comprises two aspects:

[0046] Use as a molecular tool for further researching the mechanism foramyloid formation in these illnesses (use in research laboratories and Rand D laboratories)

[0047] Potential use as a reagent for diagnosing amyloid illnesses orthe prediction of such illnesses (diagnostic market).

[0048] The invention is explained subsequently in greater detail withthe aid of an embodiment example.

[0049] NFGAIL: Production, characterisation, aggregation, testing forfibril formation, testing for cytotoxicity, testing for the inhibitoreffect on the aggregation of IAPP.

[0050] Production and Characterisation

[0051] NFGAIL was produced using current methods of fixed phase peptidesynthesis. The Wang anchor was used and the Fmoc/tBu synthesis strategywas adopted. For one 1 mMol application, 4 mMol protected amino acids, 4mMol of TBTU and 6 mMol of DIEA in DMF were used per coupling step. Thesplitting of the temporary protected groups (Fmoc) was achieved by meansof 25% piperidine in DMF and, in order to split the peptide from theanchor while simultaneously splitting the permanent protected groups ofthe side chains, 95% TFA was used (reaction time 2 hours). The pipeproduct was cleaned using preparative RP-HPLC and characterised byFAB-MS and amino acid analysis.

[0052] Aggregation and Testing for Fibril Formation

[0053] The aggregation properties were tested in 10 mM Of phosphatebutter pH 7.4. Firstly, a highly concentrated (125-250 mM) parentsolution of the peptide in DMSO was produced. The peptide is pipettedfrom the solution directly into the aggregation solution while beinggently stirred. A 5 mM peptide solution aggregated spontaneously andthus did not have an “Aggregation-Lag-Time”. On the other hand,aggregate formed only after 9.5 hrs. from a solution of 3.75 mM NFGAIL(Aggregation-Lag-Time: 9.5 hrs) (FIG. 2). Furthermore, aggregation wasaccomplished immediately, when pre-prepared NFGAIL fibrils (nucleationcentres) were added to the 3.75 mM solution (fibril concentration 0.375mM). It was shown thus, that the aggregation of these peptides operatesaccording to the so-called “nucleation dependent polymerisationmechanism”. Recently it has been considered of great importance, sincethe opinion prevails in the literature, that amyloid formation in vitroshould operate using this mechanism. After complete aggregation, thedeposit was isolated by centrifuging and examined by electron- andpolarisation- microscopy after congo red staining. In this way, thefibrillar and amyloid structure of the aggregate was confirmed (FIG. 3).

[0054] Testing for Cytotoxicity

[0055] Suspensions of the peptide aggregate (produced as described asabove) were tested by means of a Rat Insulinoma (RIN5 mf) and a humanastroglioma-cell series (HTB-14) for toxicity. The newly dissolvedpeptides were also tested for cytotoxicity. It thus be shown, that theaggregated form of NFGAIL is cyto-toxic, the soluble form of the peptidehaving no toxicity (FIG. 4).

In the claims:
 1. A peptide acting as at least one of an agonist andinhibitor of at least one of amyloid formation and cytotoxicitycomprising 3-15 amino acids and at least an active peptide sequence GA.2. The peptide according to claim 1 , comprising a maximum of 10 aminoacids.
 3. The peptide according to claim 1 , comprising an activepeptide sequence GAI.
 4. The peptide according to claim 3 , wherein thepeptide is selected from the group consisting of GAIL, FGAIL, NFGAIL,NNFGAIL, SNNFGAIL, NFGAILSS and SNNFGAILSS.
 5. The peptide according toclaim 3 , wherein the peptide is selected from the group consisting ofGAII, KGAII, NKGAII, SNKGAII, GSNKGAII, NKGAIIGL and GSNKGAIIGL.
 6. Thepeptide according to claim 1 , wherein the peptide is selected from thegroup consisting of AGAVV, AAGAVV, VYYGAVV, HVAAGAVV, AAGAVVGG andHVAAGAVVGG.
 7. The peptide according to claim 1 , wherein in the peptidesequence at least one hydrogen molecule of an amide bond is replaced bya methyl group.
 8. The peptide according to claim 7 , wherein everysecond hydrogen molecule is replaced by a methyl group.
 9. The peptideaccording to claim 2 , comprising an active peptide sequence GAI. 10.The peptide according to claim 9 , wherein the peptide is selected fromthe group consisting of GAIL, FGAIL, NFGAIL, NNFGAIL, SNNFGAIL, NFGAILSSand SNNFGAILSS.
 11. The peptide according to claim 9 , wherein thepeptide is selected from the group consisting of GAII, KGAII, NKGAII,SNKGAII, GSNKGAII, NKGAIIGL and GSNKGAIIGL.
 12. The peptide according toclaim 2 , wherein the peptide is selected from the group consisting ofAGAVV, AAGAVV, VYYGAVV, HVAAGAVV, AAGAVVGG and HVAAGAVVGG.
 13. Acomposition acting as at least one of an agonist and inhibitor of atleast one of amyloid formation and cytotoxicity in Type II diabetesmellitus comprising a peptide comprising 3-15 amino acids and an activepeptide sequence GA.
 14. The composition according to claim 13 , whereinthe peptide comprises a maximum of 10 amino acids.
 15. The compositionaccording to claim 13 , wherein the peptide comprises an active peptidesequence GAI.
 16. The composition according to claim 13 , wherein thepeptide is selected from the group consisting of GAIL, FGAIL, NFGAIL,NNFGAIL, SNNFGAIL, NFGAILSS and SNNFGAILSS.
 17. A composition acting asat least one of an agonist and inhibitor of at least one of amyloidformation and cytotoxicity in Alzheimer's disease comprising a peptidecomprising 3-15 amino acids and at least an active peptide sequence GA.18. The composition according to claim 17 , wherein the peptidecomprises an active peptide sequence GAI.
 19. The composition accordingto claim 17 , wherein the peptide is selected from the group consistingof GAII, KGAII, NKGAII, SNKGAII, GSNKGAII, NKGAIIGL and GSNKGAIIGL. 20.An agonist of at least one of amyloid formation and cytotoxicitycomprising a peptide comprising 3-15 amino acids and at least an activepeptide sequence GA.
 21. The agonist according to claim 20 , wherein theagonist acts as agonist of at least one of amyloid formation andcytotoxicity in spongiform encephalopathies.
 22. The agonist accordingto claim 21 , wherein the peptide comprises a maximum of 10 amino acids.23. The agonist according to claim 21 , wherein the peptide is selectedfrom the group consisting of AGAVV, AAGAVV, VYYGAVV, HVAAGAVV, AAGAVVGGand HVAAGAVVGG.
 24. An inhibitor of at least one of amyloid formationand cytotoxicity comprising a peptide comprising 3-15 amino acids and atleast an active peptide sequence GA.
 25. The inhibitor according toclaim 24 , wherein the inhibitor inhibits at least one of amyloidformation and cytotoxicity in spongiform encephalopathies.
 26. Theinhibitor according to claim 25 , wherein the peptide comprises amaximum of 10 amino acids.
 27. The inhibitor according to claim 25 ,wherein the peptide is selected from the group consisting of AGAVV,AAGAVV, VYYGAVV, HVAAGAVV, AAGAVVGG and HVAAGAVVGG.